Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High-throughput analysis of the plasmid bioproduction process in Escherichia coli by FTIR spectroscopy

Monitoring plasmid production systems is a lab intensive task. This article proposes a methodology based on FTIR spectroscopy and the use of chemometrics for the high-throughput analysis of the plasmid bioproduction process in E. coli. For this study, five batch cultures with different initial medium compositions are designed to represent different biomass and plasmid production behavior, with the maximum plasmid and biomass concentrations varying from 11 to 95 mg L(-1) and 6.8 to 12.8 g L(-1), respectively, and the plasmid production per biomass varying from 0.4 to 5.1 mg g(-1). After a short sample processing consisting of centrifugation and dehydration, the FTIR spectra are recorded from the collected cellular biomass using microtiter plates with 96 wells. After spectral pre-processing, the predictive FTIR spectra models are derived by using partial least squares (PLS) regression with the wavenumber selection performed by a Monte-Carlo strategy. Results show that it is possible to improve the PLS models by selecting specific spectral ranges. For the plasmid model, the spectral regions between 590-1,130, 1,670-2,025, and 2,565-3,280 cm(-1) are found to be highly relevant. Whereas for the biomass, the best wavenumber selections are between 900-1,200, 1,500-1,800, and 2,850-3,200 cm(-1). The optimized PLS models show a high coefficient of determination of 0.91 and 0.89 for the plasmid and biomass concentration, respectively. Additional PLS models for the prediction of the carbon sources glucose and glycerol and the by-product acetic acid, based on metabolism-induced correlations between the nutrients and the cellular biomass are also established.     

A study on medium chain length-polyhydroxyalkanoate accumulation in Escherichia coli harbouring phaC1 gene of indigenous Pseudomonas sp. LDC-5

AIMS: This study is mainly focused on the heterologous expression and accumulation of polyhydroxyalkanoates (PHA) in Escherichia coli. METHODS AND RESULTS: PHA synthase gene (phaC1) from indigenous Pseudomonas sp. LDC-5 was amplified by PCR and cloned in E. coli (Qiagen EZ competent cells). The recombinant E. coli was analysed and confirmed for its expression of phaC1 gene by phase contrast microscopy, Western blot analysis and spectral studies (Fourier-transform infrared spectroscopy). It was further evaluated for its accumulation in different carbon and nitrogen sources. The accumulation of PHA (3.4 g l(-1)) was enhanced in the medium supplemented with glycerol and fish peptone compared to the other carbon and nitrogen sources used in this study. CONCLUSIONS: This study would enable the reduction of cost of PHA production. SIGNIFICANCE AND IMPACT OF THE STUDY: An important part of this study is that E. coli harbouring partial phaC1 gene could accumulate medium chain length PHA significantly. The results demonstrated that the E. coli strain could be a potential candidate for the large-scale production of polymer. The conditions for the higher yield and productivity will be optimized in the next phase using fermentation studies.     

Fourier transform infrared (FTIR) spectroscopy for identifying Lactobacillus species

Abstract Fourier Transform infrared (FTIR) spectroscopy can be used to identify microorganisms. This study describes the influence of culture conditions on FTIR spectra and the discrimination of Lactobacillus species found in breweries. Fifty three Lactobacillus strains were analysed by FTIR spectroscopy and identification at the species level was correct for 94% of the strains, and at the strain level for 91% of the strains.     

Influence of plant polyphenols and medicinal plant extracts on antibiotic susceptibility of Escherichia coli

Aims: To investigate the influence of polyphenols and plant extracts on the susceptibility of Escherichia coli to antibiotics. Methods and Results: Susceptibility of E. coli to antibiotics in the presence of extracts and polyphenols was estimated by the determination of the minimum inhibitory concentrations (MICs). To study gene expression, we used strains of E. coli carrying fusions between promoters of genes katG, sodA, iucC and structural β‐galactosidase gene. Treatment with polyphenols and some plant extracts significantly decreased the antibacterial effects of antibiotics, to a larger extent, ciprofloxacin. The most remarkable protective effect was observed for the extracts of Chamerion (Epilobium) angustifolium, Filipendula vulgaris, Tanacetum vulgare and Serratula coronata. These extracts increased the MICs of ciprofloxacin by four and more times. In case of kanamycin, extracts of Artemisia austriaca and Artemisia pontica increased MICs by four and eight times, respectively. Polyphenol quercetin also caused protective effect against ciprofloxacin, increasing the MIC by four times. A positive correlation was found between protective effects of polyphenols and extracts and their antioxidant activity. Conclusion: Medicinal plant extracts and polyphenols may protect cells of E. coli against antibiotic toxicity. Significance and Impact of the Study: The results of this study may be used to enhance the efficiency of antibacterial therapies.     

Characterization of different compost extracts using Fourier-transform infrared spectroscopy (FTIR) and thermal analysis

Compost extract or “compost tea” is a liquid extract of compost obtained by mixing compost and water for a defined period of time. Compost tea contains nutrients and a range of different organisms and is applied to the soil or directly to plants with the principal aim of suppressing certain plant diseases. In addition, the application of compost tea supplies nutrients and organic matter to the soil. Thermal analysis and Fourier transform infrared spectroscopy (FTIR) are two widely applied analytical techniques for establishing the stability of compost, and although numerous studies have evaluated the capacity of compost tea to suppress plant diseases, there are no studies employing these techniques to characterize compost-tea. For the present study, 12 compost extracts were produced under varying conditions in a purpose-built reactor. Two different composts, an stable compost produced from manure and an unstable compost produced from municipal solid waste, respectively, two aeration systems (aerated and non-aerated extracts) and three temperatures (10, 20 and 30°C) were used in these experiments. The extracts were freeze-dried and subsequently analysed, together with the two composts, by means of FTIR and thermal analysis. Extracts produced from high stability compost, independently of the conditions of aeration and temperature, showed very similar results. In contrast, differences among extracts produced from the unstable compost were more noticeable. However, the different conditions of aeration and temperature during the production of the extracts only explained partially these differences, since the transformations undergone by compost over the 3 months that the experiments lasted were also reflected in the composition of the extracts. In spite of everything, extraction process favoured the degradation of easily oxidizable organic matter, which was more abundant in unstable compost. This degradation was more intense for non-aerated processes, probably due to the longer duration of these (10 days) with respect to aerated extractions (2 days). The effect of temperature was not clear in these experiments, although high temperatures could increase micro organism activity and consequently favour the degradation of easily oxidizable organic matter.     

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.     

Impact of Delftia tsuruhatensis and Achromobacter xylosoxidans on Escherichia coli dual-species biofilms treated with antibiotic agents

Recently it was demonstrated that for urinary tract infections species with a lower or unproven pathogenic potential, such as Delftia tsuruhatensis and Achromobacter xylosoxidans, might interact with conventional pathogenic agents such as Escherichia coli. Here, single- and dual-species biofilms of these microorganisms were characterized in terms of microbial composition over time, the average fitness of E. coli, the spatial organization and the biofilm antimicrobial profile. The results revealed a positive impact of these species on the fitness of E. coli and a greater tolerance to the antibiotic agents. In dual-species biofilms exposed to antibiotics, E. coli was able to dominate the microbial consortia in spite of being the most sensitive strain. This is the first study demonstrating the protective effect of less common species over E. coli under adverse conditions imposed by the use of antibiotic agents.     

一种基于代谢网络分析最小化基因组的方法及其在大肠杆菌中的应用

最小生命体的合成是合成生物学研究的重要方向。最小化基因组的同时而又不对细胞生长产生影响是代谢工程研究的一个重要目标。文中提出了一种从基因组尺度代谢网络模型出发,通过零通量反应删除及对非必需基因组合删除计算获得基因组最小化代谢网络模型的方法,利用该方法简化了大肠杆菌经典代谢网络模型iAF1260,由起始的1 260个基因简化得到了312个基因,而最优生物质生成速率保持不变。基因组最小化代谢网络模型预测了在细胞正常生长的前提下包含最少基因的代谢途径,为大肠杆菌获得最小基因组的湿实验设计提供了重要参考。     

Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France)

Over 6 years, Escherichia coli were isolated from water samples from seven Seine estuary stations, characterized by a densely populated watershed (654 isolates). Resistances of these E. coli to 16 antibiotics were determined and compared with the same resistances in E. coli isolated from a small stream (120 isolates) and from the treated effluent of the largest estuary wastewater treatment plant (123 isolates). Between 30.2% and 56.6% of the estuary isolates were resistant, whatever the station or time of sampling; of these, 60.5–80% were resistant to at least two and up to 12 antibiotics. In the three contrasting sites, resistances to tetracycline, amoxicillin and ticarcillin were the commonest. DNA was extracted from 279 estuary isolates (January 2006) and class 1, 2 and 3 integrons were detected by multiplex real-time PCR and confirmed by classic PCR. IntI1 and intI2 genes were found in 11% of isolates. No intI3 gene was detected. The variable regions of the class 1 and 2 integrons sequenced contained predominantly gene cassettes aadA and dfr . However, for slightly over half of the E. coli isolates exhibiting the class 1 integron, the variable region could not be amplified, because part of the 3' conserved sequence was missing.     

Salt stress effects on the central and carnitine metabolisms of Escherichia coli

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.     

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil

Enterotoxigenic Escherichia coli (ETEC) strains have been implicated as important etiological agents of diarrheal disease, especially in developing countries. This group of microorganisms has been associated with a diverse range of genotypic and phenotypic markers. In the present study, 21 ETEC isolates previously defined according to the toxigenic genotypes, were characterized on the basis of O:H typing, cell adherence patterns, and colonization factors (CFs) antigens. Genetic diversity was investigated by random amplification polymorphic DNA (RAPD-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE). LT-I probe-positive isolates belonged to serotypes ONT:HNT, O7:H24, O48:H21, O88:H25, O148:H28, O159:H17 and O159:H21. ST-h probe-positive isolates belonged to serotypes O159:H17, O148:H28 and O6:H-. Serotypes O148:H28, O159:H17 and O6:H- were associated with the CS6, CFA/I and CS1 CS3 antigens, respectively. Most ETEC strains exhibited a diffuse pattern of adherence to cultured epithelial cells. In general, phenotypic and genotypic characteristics correlated well. RAPD-PCR, PFGE and MLEE showed reproducibility and good discriminatory potential. The application of molecular typing systems allowed the detection of significant diversity among the isolates, indicating a non-clonal origin and revealing intra-serotype variation overlooked by classical epidemiological approaches. The phenotypic and genotypic diversity observed lead us to recommend the use of different typing systems in order to elucidate the epidemiology of ETEC infection.     

The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins

The Fe atom in the bimetallic active site of [NiFe]-hydrogenases has one CO and two cyanide ligands. To determine their metabolic origin, [NiFe]-hydrogenase-2 was isolated from Escherichia coli grown in the presence of L-[ureido-(13)C]citrulline, purified and analyzed by infrared spectroscopy. The spectra indicate incorporation of (13)C only into the cyanide ligands and not into the CO, showing that cyanide and CO have different metabolic origins. After growth of E. coli in the presence of (13)CO only the CO ligand was labelled with (13)C. Labelling did not result from an exchange of the intrinsic CO ligand with the exogenous CO.     

透明颤菌血红蛋白的表达及对基因工程菌的影响

利用已克隆的透明颤菌(Vitreoscilla)血红蛋白基因(vgb),构建了一批复制类型和抗生标记不同的vgb表达载体,并就vgb基因表达及其对几种基因工程大肠杆菌的影响进行了初步研究。实验证明vgb基因的表达具有氧调控特性,在溶氧水平下跌至20％饱和度时迅速合成。Vgb基因的表达产物(Vitreoscilla Hemoglogin,VHb)可促进青霉素酰化酶和TNF、IL-2等基因工程菌在低氧条件下细胞生长和产物表达的状况,由于vgb基因的表达降低了细胞对氧的敏感程度,可望运用它来改善发酵过程中溶氧控制裕度。这些实验结果预示着vgb基因在耗氧生物过程中,如抗生素工业和基因工程菌高密度发酵,有着良好的应用前景。     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Near infrared spectroscopy, cluster and multivariate analysis hyphenated to thin layer chromatography for the analysis of amino acids

Summary. A method based on near-infrared spectroscopy (NIRS) was developed for the rapid and non-destructive determination and quantification of solid and dissolved amino acids. The statistical results obtained after optimisation of measurement conditions were evaluated on the basis of statistical parameters, Q-value (quality of calibrations), R², standard error of estimation (SEE), standard error of prediction (SEP), BIAS applying cluster and different multivariate analytical procedures. Experimental optimisation comprised the selection of the highest suitable optical thin-layer (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mm), sample temperature (10–30 °C), measurement option (light fibre, 0.5 mm optical thin-layer; boiling point tube; different types of cuvettes) and sample concentration in the range between 100 and 500 ppm. Applying the optimised conditions and a 115-QS Suprasil^? cuvette (V = 400 μl), the established qualitative model enabled to distinguish between different dissolved amino acids with a Q-value of 0.9555. Solid amino acids were investigated in the transflectance mode, allowing to differentiate them with a Q-value of 0.9155. For the qualitative and quantitative analysis of amino acids in complex matrices NIRS was established as a detection system directly onto the plate after prior separation on cellulose based thin-layer chromatography (TLC) sheets employing n-butanol, acetic acid and distilled water at a ratio of 8:4:2 (v/v/v) as an optimised mobile phase. Due to the prior separation step, the established calibration curve was found to be more stable than the one calculated from the dissolved amino acids. The found lower limit of detection was 0.01 mg/ml. Finally, this optimised TLC-NIRS method was successfully applied for the qualitative and quantitative analysis of L-lysine in apple juice. NIRS is shown not only to offer a fast, non-destructive detection tool but also to provide an easy-to-use alternative to more complicated detection methods such as mass spectrometry (MS) for qualitative and quantitative TLC analysis of amino acids in crude samples.     

Isolation,molecular characterization and antibiotic resistance of Shiga Toxin–Producing Escherichia coli (STEC) from buffalo in India

In total, 363 Escherichia coli were isolated from 165 faecal samples of healthy buffaloes in West Bengal, India. Twenty‐four of these isolates (6·61%) were found to carry at least one gene characteristic for Shiga toxin–producing Escherichia coli (STEC). These STEC strains belonged to 13 different O‐serogroups. The stx₁ gene was present in 23 (95·8%) of total STEC isolates, whereas 20 (83·3%) STEC isolates carried the gene stx_2. Twelve strains of E. coli (50% of total STEC isolates) possessed enterohaemolysin (ehxA) gene in combination with others. Fourteen (58·33%) isolates found to possess saa gene. However, no E. coli was detected harbouring gene for intimin protein (eaeA). Of 23 stx₁‐positive isolates, seven (30·43%) were positive for genes of the stx_1C subtype. Of the 20 isolates with the stx₂ gene, 25% (5/20) possessed stx_2C and 10% (2/20) possessed stx_2d gene. The phylogenetic analysis after RAPD of STEC strains revealed six major clusters. The isolated STEC strains were resistant most frequently to erythromycin (95·83%), cephalothin (62·5%), amikacin (54·17%), kanamycin (45·83%) and gentamicin (41·67%) group of antibiotics. No ESBL‐producing (bla_CTXM, bla_TEM, bla_SHV) or quinolone resistance gene (qnrA) was detected in the STEC isolates.

Significance and Impact of the Study

The buffaloes from different districts of West Bengal, India, are important reservoir of multidrug‐resistant Shiga toxin–producing Escherichia coli (STEC). India is home to more than 56% of world buffalo population, traditionally raised by farmers. So, there is a major risk of transmission of STEC among the human population of this part of the globe. However, there is no prevalence study of STEC from healthy or diarrhoeic buffalo in India. The present study reports for the first time in India about isolation, molecular characterization and antibiotic resistance pattern of STEC in healthy buffaloes.     

Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique Resources for Biological Research

Based on the genomic sequence data of Escherichia coli K-12strain, we have constructed a complete set of cloned individualgenes encoding Histidine-tagged proteins with or without GFPfused for functional genomic analysis. Each clone encodes aprotein of predicted ORF attached by Histidines and seven spaceramino acids at the N-terminal end, and five spacer amino acidsand GFP at the C-terminal end. SfiI restriction sites are generatedat both the N- and C-terminal boundaries of ORF upon cloning,which enables easy transfer of ORF to other vector systems bycutting with SfiI. Expression of cloned ORF is under the controlof an IPTG-inducible promoter, which is strictly repressed bylacI^q repressor gene product. The set of cloned ORFs describedhere should provide unique resources for systematic functionalgenomic approaches including (i) construction of DNA microarray,(ii) production and purification of proteins, (iii) analysisof protein localization by monitoring GFP fluorescence and (iv)analysis of protein–protein interaction.     

Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.